M1-2 and M2-3 cells were established from mouse tongue tumor specimens induced by 4-NQO and arecoline. M1-2 and M2-3 cells partially developed tumors after subcutaneous injection in immune-compromised mice. In contrast, M1-2 and M2-3 cells failed to develop any tumor by orthotopic inoculation of syngeneic mice. By in vitro selection, M1-2sph and M2-3sph cells were established and only M1-2sph cells successfully developed tumors in syngeneic mice. M1-2sph and M2-3sph cells showed the higher activities of cell growth and sphere formation than their parental cells. The higher Klf4 and Nanog expression was detected only in M1-2sph cells. M1-2sph and M2-3sph cells showed the higher capabilities of migration and invasion than their parental cells. The epithelial mesenchymal transition (EMT) was occurred only in M1-2sph cells. The higher Twist and Slug expression was detected only in M1-2sph cells. The profiling of p ERK1/2 activation was changed between M1-2 and M1-2sph cells but not between M2-3 and M2-3sph cells. The relationship between ERK1/2 activation and occurrence of EMT and sphere formation was under investigation.